Recent cases of human infection with H5N1 avian influenza viruses remind us of the serious threat future influenza pandemics pose to public health and the global economic infrastructure. To date, there have been more than 400 human infections with highly pathogenic H5N1 influenza viruses, resulting in greater than 250 deaths. The 60% mortality rate associated with human H5N1 influenza infection is remarkable when compared to the <0.1% mortality rate of contemporary seasonal influenza viruses. This dramatic disparity highlights a critical unanswered question: Why are highly pathogenic avian influenza (HPAl) viruses so deadly? Viral RNAs generated during the influenza life cycle contain conserved molecular epitopes that stimulate the production of cytokines and chemokines through host viral pattern recognition receptors. A number of studies have revealed elevated levels of inflammatory cytokines and chemokines in HPAI-infected lungs, suggesting that dysregulated host immune responses are central to the tissue damage, respiratory distress, and mortality associated with HPAl infection. Our laboratory reported that a lysine residue at position 627 of the HPAl PB2 polymerase subunit confers increased replicative efficiency and is a major virulence determinant for HPAl viruses. This suggests a direct connection between the generation of immune-stimulating influenza vRNA and the pathological host immune response induced by HPAl infection. The long-term objective of this project is to define the molecular pathways that connect H5N1-type HPAl virus replication with the severe immunopathology associated with infection. Aim 1 will use flow cytometric approaches to define the patterns of immune cell recruitment to the mouse lung during HPAl infection. In Aim 2, we will evaluate mice lacking components of toll-like and RlG-1-like receptor pathways to determine the contribution of viral pattern recognition pathways to immune cell recruitment and cytokine production during HPAl infection. In Aim 3, we will evaluate the impact of viral replication efficiency on immune cell recruitment kinetics and cytokine responses by comparing a highly pathogenic PB2-627K H5N1 virus with a PB2-627E virus counterpart. Relevance: Highly pathogenic avian influenza viruses continue to circulate and pose a major risk for a new influenza pandemic. It is unclear how highly pathogenic H5N1 viruses induce aberrant host immune responses and subsequent respiratory failure. By studying fundamental aspects of immunological responses to H5N1 influenza viruses, we will contribute to the rational design of vaccines and the development of new drugs for chemoprophylaxis.